Gcsfa and Gcsfb expand definitive neutrophils and mφs in the zebrafish embryo. (A) Fluorescence images of 72 hpf mpx:GFP transgenic zebrafish injected with PBS (mock) and either in vitro–transcribed gcsfa or gcsfb mRNA. Fluorescent images taken on a Leica DMI-6000 inverted fluorescent scope with a Hamamatsu Photonics Orca 3CCD color digital camera at ×50 and processed by Volocity (Perkin Elmer) and Photoshop (Adobe Systems) software. Images shown are 2 images stitched together. (B) Numbers of mpx:GFP⁺ cells at 72 hpf (y-axis) after injection of PBS (mock, circles) and gcsfa (squares) or gcsfb (triangles) mRNA at the single-cell stage of development. Mean (dashed red line) with 95% confidence interval (red error bars) and level of statistical significance. *P < .0001. (C) Percentage of mpeg1:GFP⁺ cells at 72 hpf (y-axis) after injection of PBS (mock, circles) and gcsfa (squares) or gcsfb (triangles) mRNA. Each data point corresponds to 5 embryos pooled together before digestion and flow cytometry. Mean (dashed red line) with 95% confidence interval (red error bars) and level of statistical significance. *P < .003; **P < .0006; ***P < .0001. (D) At 72 hpf, mφs and neutrophils express gcsfr. qRT-PCR analysis of gcsfr in FACS-isolated macrophages (mpeg1:GFP⁺ cells at 72 hpf), neutrophils (mpx:GFP⁺ cells at 72 hpf), and adult zebrafish kidney (Whole Kidney) shown for reference. Levels are relative to the housekeeping gene ef1α. All samples are at least biological duplicate preparations. Bars represent the mean, and error bars represent SEM. *P < .0001.